PROJECTSUMMARY Foodallergy(FA)affects8%ofchildrenand5%ofadultsintheU.S.,and30%ofthosehaveclinical reactivitytomultiplefoods.In2independentphase1clinicaltrials,weshowedthatsimultaneousoral desensitization(D)tomultiplefoodallergens(multi-OIT)issafeandfeasible,andcanbeachievedin6-9 monthswithanti-IgEadjunctivetherapy.PolarizationofnaveTcellsintoIL-4-secretingTh2cellsisthefirst stepleadingtoallergicresponses.Thus,understandinghowmodulationofTcellresponsescanleadtoD orsustainedunresponsiveness(SU)duringsuccessfulOITiscritical.WeproposetomonitorTcellsusing innovativetechnologiesin:(1)eachofthecohortsproposedinProject1(i.e.,multi-FAparticipants(n=60) treatedwithmulti-OIT+/-omalizumabordupilumabwhodevelopD[definedasapositivefoodchallenge reactionaftera6weekwithdrawalofOIT]vs.SU[definedasanegativefoodchallengereactionafter withdrawalofOIT]totherespectiveallergensintheirmulti-OIT);?(2)longtermfollowupstudiesof>240 participantsonOIT;?and(3)GIbiopsiesobtainedovertimeinOITparticipants).InProject3,wewill investigatewhetherchangesinparticipants?Tcellsubpopulationscanidentifymarkerspredictiveofclinical outcomes.Weparticularlywillfocusonchangesinallergen-specificTh2cellsinthosewhoexhibitfavorable responsestoOIT.BycharacterizingandquantifyingthemodulationofTcellphenotypeandfunction associatedwithvariousmulti-OIToutcomes,wewillidentifyTcellsignaturesofSUinmulti-FAparticipants. Ourmainhypothesesarethatsuccessfulmulti-OITwill:(1)reprogramtotalandallergen-specificTh2cells toTh1and/orTregsubtype,(2)replaceallergen-specificTh2cellsbyTh1andTregsubtype,and/or(3) expandallergen-specificcloneswithdiversephenotypeandfunction,potentiallyoverridingtheeffectsof Th2cells.WespeculatethatstableepigeneticchangesinIL4,IL10,IFN?and/orFOXP3genesmediatethe anticipatedshiftfromTh2phenotype,contributingtoSU.Totestthesehypotheses,weproposeto:(Aim1) Characterizetheimmunophenotypicandfunctionalchangesinducedbymulti-OITintotalandallergen- specificTcells;?(Aim2)UseMHCclassIImultimerstosortallergen-(peanut/milk/cashew)specificsingle cellsandperformtargetedRNA-seqtoinvestigatetheirmolecularsignaturesandclonalancestryatsingle cellresolution;?and(Aim3)Quantifyepigeneticchanges(i.e.,methylationofCpGislands)inkeygenes (i.e.,FOXP3,IL4,IFN?,IL10)toassesspossiblelinksbetweengenemethylation,andthusexpressionof thesegenes,andfavorableOITclinicaloutcomes.Ifweachievetheseaims,weexpectourresultswillboth providenewinsightsintothemechanismsunderlyingsuccessfulclinicaloutcomesinmulti-OITandimprove understandingoftheimmunechangesthatcancontributetosuccessfuloutcomesinOIT.